Recombinant human erythropoietin mutants and therapeutic methods employing them

ABSTRACT

DNA encoding modified, secretable erythropoietin proteins whose ability to regulate the growth and differentiation of red blood cell progenitors are different from the wildtype recombinant erythropoietin and to methods of modifying or altering the regulating activity of a secretable erythropoietin and using modified secretable erythropoietin proteins.

GOVERNMENT SUPPORT

The invention described herein was supported in whole or in part byGrant No. 38841 from the National Institutes of Health and Grant No.N00014-90-1847 from the U.S. Navy.

RELATED APPLICATIONS

This application is a continuation-in-part application of U.S. Ser. No.08/113,080, filed Aug. 26, 1993, now abandoned, which is acontinuation-in-part application of U.S. Ser. No. 07/920,810, filed Jul.28, 1992, also now abandoned. The teachings of these relatedapplications are incorporated herein by reference.

BACKGROUND

The glycoprotein hormone erythropoietin regulates the growth anddifferentiation of red blood cell (erythrocyte) progenitors. The hormoneis produced in the fetal liver and adult kidney. Erythropoietin inducesproliferation and differentiation of red blood cell progenitors throughinteraction with receptors on the surface of erythroid precursor cells.

Several approaches have been employed to identify those features of theprotein that are relevant to its structure and function. Examination ofthe homologies among the amino acid sequences of erythropoietin proteinsof various species has demonstrated several highly conserved regions(McDonald, J. D., et al., Mol. Cell. Biol. 6:842-848 (1986)).

Oligonucleotide-directed mutagenesis has been used to prepare structuralmutants of erythropoietin, lacking specific sites for glycosylation.Studies indicate that N-linked carbohydrates are important for properbiosynthesis and/or secretion of erythropoietin. These studies also showthat glycosylation is important for in vivo, but not in vitro,biological activity. (Dube, S., et al., J. Biol. Chem. 263:17516-17521(1988); Yamaguchi, K., et al., J. Biol. Chem. 266:20434-20439 (1991);Higuchi, M., et al., J. Biol. Chem. 267:7703-7709 (1992)).

Studies with monoclonal anti-peptide antibodies have shown that theamino terminus and the carboxy-terminal region (amino acids 152-166) oferythropoietin may be involved with biological activity. It has alsobeen demonstrated that antibodies to amino acids 99-119 and 111-129block the hormone's biological activity, apparently by binding to twodistinct non-overlapping domains (99-110 and 120-129). (Sytkowski, A. J.and Donahue, K. A., J. Biol. Chem. 262:1161-1165 (1987)). Thus, it washypothesized that amino acids 99-129 were important in the formation ofa functional region involved in receptor recognition, either throughforming a necessary component of the protein's tertiary structure orthrough direct participation in receptor binding, or both.

Preliminary experiments suggested that alterations in localizedsecondary structure within the 99-129 region resulted in inactivation oferythropoietin. Therefore, a possible structural role for amino acids99-129 has been postulated. Recently, a series of experiments indicatedthat amino acids 99-110 (Domain 1) play a critical role in establishingthe biologically active conformation of human erythropoietin. (Chern,Y., et al., Eur. J. Biochem. 202:225-229 (1991)).

These Domain 1 mutants, in which a group of three amino acids wasdeleted and replaced by two different amino acids, were found to bebiologically inactive. Furthermore, these mutations in Domain 1inhibited the secretion of the mutant erythropoietin into cell culturemedium. (Chern, Y., et al., Eur. J. Biochem. 202:225-229 (1991)).Inhibition of secretion in mammalian cells is consistent with a profoundstructural change of the polypeptide hormone. Profound structuralchanges could significantly affect the ability of the hormone tointeract with its cognate receptor. Thus, these mutant erythropoietinpolypeptides are not suitable for elucidating the structure/functionrelationship that exists between erythropoietin and its cellularreceptor. Nor are these mutants suitable erythropoietin antagonists foruse, for example, in therapeutic treatment of polycythemias, or overproduction of erythropoietin. Thus, it would be beneficial to preciselydetermine which amino acids are critical to the erythropoietinpolypeptide to maintain a stable, biologically active conformation whichretains its secretable properties and its ability to bind to theerythropoietin receptor.

Moreover, the precise determination of critical amino acid residueswould be useful to alter the biological activity of erythropoietin,either decreasing or increasing one or more biological properties of theprotein.

SUMMARY OF THE INVENTION

The present invention relates to isolated DNA encoding mutatederythropoietin proteins which have altered biological activity, yetretain their secretable properties (i.e., secretable erythropoietinproteins). That is, the present invention relates to isolated DNAencoding secretable erythropoietin proteins which have at least oneamino acid residue in Domain 1 which differs from the amino acid residuepresent in the corresponding position of wildtype erythropoietin andwhich have altered ability to regulate the growth and differentiation ofred blood cell progenitors. Domain 1 of the mutants described hereinrefers to the amino acids which correspond to amino acids 99-110 (SEQ IDNO: 1) of the wildtype recombinant erythropoietin. Altered ability isdefined as ability different from that of the wildtype recombinanterythropoietin ability to regulate the growth and differentiation of redblood cell progenitors. As used herein, altered ability to regulate thegrowth and differentiation of red blood cell progenitor cells refers tobiological activity different from wildtype recombinant erythropoietinactivity (i.e., altered biological activity relative to wildtyperecombinant erythropoietin activity). The mutated erythropoietinproteins of the present invention can be secreted in homologous andheterologous expression systems. For example, the mutated erythropoietinproteins of the present invention can be secreted in mammalian,bacterial or yeast expression systems.

The present invention also relates to the modified secretable mutanterythropoietin proteins encoded by the isolated DNA described above.These modified secretable erythropoietin proteins have alteredbiological activities. For example, the modified secretable mutanterythropoietin may have decreased ability relative to wildtypeerythropoietin protein to regulate growth and differentiation of redblood cell progenitor cells. As used herein, decreased ability toregulate growth and differentiation of red blood cell progenitor cellsis also referred to as decreased biological activity relative towildtype erythropoietin activity. Wildtype erythropoietin activity isalso referred to herein as biological activity of wildtypeerythropoietin. Alternately, a modified secretable mutant erythropoietinprotein described herein may exhibit increased heat stability relativeto wildtype erythropoietin protein.

The modified erythropoietin proteins described herein comprise an aminoacid sequence with at least one amino acid residue different from theamino acid residue present at the corresponding position in Domain 1 inthe wildtype erythropoietin. These erythropoietin proteins are referredto as modified secretable human recombinant erythropoietin proteinshaving altered ability (i.e., decreasing or enhancing ability) relativeto wildtype erythropoietin protein to regulate the growth anddifferentiation of red blood cell progenitors.

The term modified, as used herein, includes substitution of a differentamino acid residue, or residues, as well as deletion or addition of anamino acid residue, or residues.

Until the present invention, mutations within the erythropoietinsequence which result in the alteration of biological activity have alsofrequently resulted in a concurrent loss of secretability of the proteinfrom transfected cells. This loss of secretability is consistent with aloss of structural integrity. (Boissel, J-P. and Bunn, H. F., "TheBiology of Hematopoiesis", pp. 227-232, John Wiley and Sons, New York(1990)). Now, the sites critical to the maintenance of a stable,biologically active conformation have been identified by means ofoligonucleotide-directed mutagenesis and have been found to occur inDomain 1 (amino acids 99-110) (SEQ ID NO: 1) of human recombinanterythropoietin. Modifications of the wildtype erythropoietin have beenmade and the encoded erythropoietin proteins have been expressed. Theresulting mutant erythropoietin proteins described herein have alterederythropoietin regulating activity, as demonstrated in theart-recognized bioassay of Krystal, G., Exp. Hematol. 11:649-660 (1983).Activity of the resulting erythropoietin proteins has also beenevaluated by commercially available radioimmunoassay protocols.

In particular, the arginine 103 site is essential for erythropoietinactivity. As shown herein, replacement of the arginine 103 by anotheramino acid results in a modified erythropoietin with significantlydecreased biological activity relative to wildtype erythropoietinactivity. Modifications at this site, as well as other sites withinDomain 1, can similarly be made to enhance regulating activity, as wellas to decrease, or reduce regulating ability.

The modified secretable erythropoietin proteins described herein provideuseful reagents to further elucidate the structure/function relationshipof erythropoietin and its cellular receptor.

Such modified secretable erythropoietin proteins with altered regulatingability can also be used for therapeutic purposes. For example, modifiederythropoietin proteins with enhanced biological activity would be amore potent therapeutic, therefore requiring a lower effective dose orless frequent administration to an individual. Erythropoietin proteinswith decreased biological activity that still retain their structuralintegrity and bind to their cognate receptor would be useful to decreasegrowth and differentiation of red blood cell precursors in certainleukemias and polycythemias. Furthermore, an erythropoietin protein thatselectively triggers only certain events within the red blood cellprecursor cell would be useful in treating various hematologicalconditions.

Further, it is expected that modified secretable mutant erythropoietinproteins with increased heat stability relative to wildtypeerythropoietin proteins would have a longer plasma half-life relative towildtype erythropoietin proteins. Thus, such modified erythropoietinproteins with increased heat stability can be useful therapeutically.For example, modified secretable mutant erythropoietin proteins withincreased heat stability would be especially important in patients witha fever and/or experiencing an increased metabolic state.

The present invention also relates to methods of modifying or alteringthe regulating activity of a secretable erythropoietin protein.

This invention further relates to pharmaceutical compositions comprisingan effective amount of modified secretable human recombinanterythropoietin in a physiologically acceptable carrier.

The present invention also relates to a method of evaluating a substancefor ability to regulate growth and differentiation of red blood cellprogenitor cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of the in vitro mutagenesis protocol.WT=wildtype erythropoietin. Shown are SEQ ID NOS: 2/3 respectively.

FIG. 2 depicts the structure of expression vector pSV-2-erythropoietin.

FIG. 3 is a graphic representation of the specific activities of ninemutant erythropoietin proteins.

FIG. 4 is a graphic representation of the results of monoclonal antibodyprecipitation of the mutant erythropoiein proteins.

FIG. 5 is a graphic representation of the activity of heat-denaturedwildtype erythropoietin as measured by radioimmunoassay (▪) and theKrystal bioassay ().

FIGS. 6A-6H are graphic representations of the activity of the 103mutant erythropoietin proteins as measured by radioimmunoassay (▪) andthe activity of wildtype erythropoietin ().

FIG. 6A shows the activity of R103A. FIG. 6B shows the activity ofR103D. FIG. 6C shows the activity of R103K. FIG. 6D shows the activityof R103E. FIG. 6E shows the activity of R103N. FIG. 6F shows theactivity of R103Q. FIG. 6G shows the activity of R103H. FIG. 6H showsthe activity of R103L.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the identification of amino acidresidues of the erythropoietin polypeptide which are critical for itsbiological activity and secretable properties. These sites have beenprecisely defined through oligonucleotide-directed mutagenesis and usedto create mutant human recombinant erythropoietin proteins which arealtered by one, or more, amino acid substitutions and thus differ fromwildtype erythropoietin.

Identification of Amino Acid Residues of Human RecombinantErythropoietin Critical for Biological Activity

Previously, anti-peptide antibodies to several hydrophilic domains ofthe erythropoietin molecule had demonstrated that antibodies to aminoacids 99-110 (Domain 1) and 111-129 (Domain 2) block the hormone'sbiological activity. Binding of the antibody to a portion of theerythropoietin molecule that participated in receptor recognition wouldblock such recognition, thereby neutralizing erythropoietin's biologicalactivity. (Sytkowski, A. J. and Donahue, K. A., J. Biol. Chem.262:1161-1165 (1987)).

A series of mutants across the 99-129 region was produced bysequentially replacing three amino acids with Glu-Phe. Mutations inamino acid residues 99-110 caused a profound structural change whichinhibited secretion of the mutant erythropoietin after biosynthesis.(Chern, Y., et al., Eur. J. Biochem. 202:225-229 (1991)). To preciselyidentify the amino acid site, or sites, critical for receptorrecognition and biological activity, amino acids 100-109 were studied byalanine scanning mutagenesis, as described in detail in Example 1.

Briefly, human recombinant erythropoietin cDNA (Powell, J. W., et al.,Proc. Natl. Acad. Sci. USA 83:6465-6469 (1986)) was inserted into thePhagemid vector pSELECT (Promega Corp., Madison, Wis.) which containstwo genes for antibiotic resistance. One of these genes, specific fortetracycline resistance is always functional, while the other, specificfor ampicillin resistance, has been inactivated. The single-strandedtemplate for the mutagenesis reaction was prepared by growing culturesof bacteria transformed with the Phagemid and infected with a helperphage. The resulting single-stranded DNA was isolated.

Two oligonucleotides were annealed to this recombinant ssDNA template.The first oligonucleotide was an ampicillin repair oligo designed toconvert the vector to ampicillin resistance and the secondoligonucleotide was a mutagenic oligo designed to change a portion ofthe erythropoietin cDNA sequence.

Subsequently, the mutant second strand was synthesized in vitro using T4DNA polymerase and ligated. This DNA was then transformed into a repairminus strain of E. coli and these cells were grown in the presence ofampicillin. The phagemid was then harvested and a second round oftransformation was carried out and mutants were selected on ampicillinplates. This resulted in the production of a double stranded phagemidcontaining both the ampicillin resistance gene and the mutatederythropoietin cDNA.

FIG. 1 shows the region of the erythropoietin cDNA encoding amino acids96-113 (SEQ ID NO: 2) and the corresponding wildtype erythropoietin DNAsequence encoding amino acids 96-113 (SEQ ID NO: 3). The column ofnumbers on the left hand side of FIG. 1 indicates the amino acidsubstitution. The only amino acid residue substitutions made were asindicated. The remainder of the human recombinant erythropoietin DNAsequence was not altered. (The remaining, unaltered human recombinantDNA sequence is not shown.) Thus, for example, 100A (SEQ ID NO: 4)indicates that amino acid 100, normally a serine residue, was replacedby alanine, 101A (SEQ ID NO: 5) indicates that glycine 101 was replacedby alanine, and so forth (SEQ ID NOS: 6-16).

Some sites were mutated more than once. For example, amino acid 103 wasmutated twice. The first mutation was the substitution of alanine forarginine 103 (SEQ ID NO: 7) and the second substitution was asparticacid for arginine (SEQ ID NO: 8).

Two double mutants were also produced, 108A/l13R (SEQ ID NO: 12) and109A/113R (SEQ ID NO: 13). In these two instances, amino acids 108 and109 were each substituted with alanine in the second mutation and thereplacement of glycine 113 with arginine was introduced. The changes innucleotide sequence in each mutagenic oligo are indicated in FIG. 1 andTable I (SEQ ID NOS: 4-22). In Table I, the underlined nucleotides arethose which differ from the wildtype erythropoietin sequence. A silentmutation designed to introduce a restriction site, Hinf I, allowingconvenient initial screening for mutated erythropoietin cDNAs, was alsointroduced.

In addition, two mutants in the region of the erythropoietin cDNAencoding amino acids 1-26 (the aminoterminus region) were produced. Inthese two instances, amino acid 14, normally an arginine, was replacedeither by alanine (14A) or aspartic acid (14D).

Each mutated erythropoietin cDNA was identified by restriction analysis,using standard laboratory protocols, and its structure was confirmed byDNA sequencing. The mutated erythropoietin cDNA was then inserted intothe expression vector pSV-2 (FIG. 2) using standard laboratorytechniques. (Mulligan, R. C., et. al. Nature 277:108-114 (1979);Maniatis, T., et al., "Molecular Cloning: A Laboratory Manual", ColdSpring Harbor Laboratories, New York (1982)).

As described in detail in Example 2, COS-7 cells were transfected withthe pSV-2-erythropoietin constructs. After three days, the supernatantmedium was harvested and the biological activity of the mutanterythropoietin proteins and wildtype erythropoietin was measured by theKrystal bioassay (Krystal, G., Exp. Hematol. 11:649-660 (1983)).Briefly, the bioassay of Krystal measures the effect of erythropoietinon intact mouse spleen cells. Mice were treated with phenylhydrazine tostimulate production of erythropoietin-responsive red blood cellprogenitor cells. After treatment, the spleens were removed, intactspleen cells were carefully isolated and incubated with various amountsof wildtype erythropoietin or the mutant erythropoietin proteinsdescribed herein. After an overnight incubation, ³ H thymidine was addedand its incorporation into cellular DNA was measured. The amount of ³ Hthymidine incorporation is indicative of erythropoietin-stimulatedproduction of red blood cells via interaction of erythropoietin with itscellular receptor. The concentration of mutant erythropoietin protein,as well as the concentration of wildtype erythropoietin, was quantifiedby competitive radioimmunoassay (Incstar, Stillwater, Minn.). Specificactivities were calculated as international units measured in theKrystal bioassay divided by micrograms as measured as immunoprecipitableprotein by RIA. Both assays used wildtype recombinant humanerythropoietin standardized against the World Health Organization SecondInternational Reference Standard preparation.

Two sets of experiments were performed in order to determine thespecific biological activities of these mutant erythropoietin proteins.Specific activities of nine of the mutant erythropoietin proteins (SEQID NOS: 4-13) assayed in the first set of experiments are shown in FIG.3. As shown in FIG. 3, the specific activities are presented as apercent of the wildtype erythropoietin activity for each mutanterythropoietin. The amino acid replaced by alanine is indicated alongthe horizontal axis. Table I also shows the specific activities of thenine mutant erythropoietin proteins (SEQ ID NOS: 4-13) as well as nineadditional mutant erythropoietin proteins (SEQ ID NOS: 14-22) againassayed in the first set of experiments. The specific activity noted inTable I is also that activity relative to wildtype erythropoietin'sactivity, which is set at 100%.

As shown in Table I, substitution of alanine for serine 104 decreasedactivity to approximately 16% of wildtype erythropoietin (SEQ ID NO:14). Substitution of alanine for leucine 105 (SEQ ID NO: 9) reduced theactivity to approximately 44 percent of wildtype erythropoietin.Substitution of alanine for leucine 108 (SEQ ID NO: 15) reduced theactivity to approximately 37% of wildtype erythropoietin.

                                      TABLE I                                     __________________________________________________________________________    ALANINE SCANNING MUTAGENESIS OF AMINO ACIDS                                   100-109 OF ERYTHROPOIETIN                                                                                                      SPECIFIC                                                                              SEQ                  MUTANT   OLIGONUCLEOTIDE                         ACTIVITY                                                                              ID                   __________________________________________________________________________                                                             NO:                  S100A    GGATAAAGCCGT CGCTGGCCTTCGCAGCCTCAC GACTCTGCTTCGGG                                                                     107.9%  4                    G101A    GCCGTCAGTG CCCTTCGCAGCCTCAC GACTCTGCTTCGGG                                                                            126.8%  5                    L102A    GCCGTCAGTGGC GCTCGCAGCCTCACC            93.3%   6                    R103A    CGTCAGTGGCCTT GCCAGCCTCAC GACTCTGCTTCGG 0.0%    7                    R103D    CGTCAGTGGCCTT GACAGCCTCAC GACTCTGCTTCGG 0.0%    8                    L105A    GGCCTTCGCAGC GCCAC GACTCTGCTTCGGG       44.0%   9                    T106A    GCCTTCGCAGCCTC GC GACTCTGCTTCGGGC       76.9%   10                   T107A    CGCAGCCTCACC GCTCTGCTTCG AGCTCTGCGAGCC  86.6%   11                   L108A/G113R                                                                            GCCTCACCACT GCCTTCG AGCTCTG CGAGCC      77.3%   12                   L109A/G113R                                                                            CCTCACCACTCTG GCTCGGGCTCTGCG            84.7%   13                   S104A    GTGGCCTTCGC GCCCTCAC GACTCTGCTTC        16.3%   14                   L108A    CCTCACCACT GCGCTTCGAGCTCTGGGAGC         36.9%   15                   L109A    CCTCACCACTCTG GCTCGGGCTCTGGG            70.2%   16                   R103N    CGTCAGTGGCCTT AACAGCCTCAC GACTCTGCTTCGG 0.0%    17                   R103E    CGTCAGTGGCCTT GAGAGCCTCAC GACTCTGCTTCGG 0.0%    18                   R103Q    CGTCAGTGGCCTTC AGAGCCTCAC GACTCTGCTTCGG 0.0%    19                   R103H    CGTCAGTGGCCTTC ACAGCCTCAC GACTCTGCTTCGG 0.0%    20                   R103L    CGTCAGTGGCCTTC TCAGCCTCAC GACTCTGCTTCGG 0.0%    21                   R103K    CGTCAGTGGCCTG AAGAGCCTCAC GACTCTGCTTCGG 10.2%   22                   __________________________________________________________________________

To further characterize the muteins obtained by substitution of the 103arginine amino acid residue (SEQ ID NOS: 7, 8 and 17-22), a second setof experiments with COS-7 cells transfected as described in Example 2with the pSV-2-erythropoietin mutant constructs encoding these muteinswas performed. The supernatant medium was again harvested after threedays and the biological activity of the mutant erythropoietin proteinswas measured by the Krystal bioassay, the concentration of mutanterythropoietin protein was quantified by competitive radioimmunoassay(Incstar, Stillwater, Minn.) and specific activities (shown in Table II)were calculated as international units measured in the Krystal bioassaydivided by micrograms as measured as immunoprecipitable protein by RIA.

                                      TABLE II                                    __________________________________________________________________________    MUTAGENESIS OF AMINO ACID                                                     Arg 103 OF ERYTHROPOIETIN                                                                                          SPECIFIC                                                                             SEQ                               MUTANT                                                                              OLIGONUCLEOTIDE                ACTIVITY                                                                             ID NO:                            __________________________________________________________________________    R103A CGTCAGTGGCCTT GCCAGCCTCAC GACTCTGCTTCGG                                                                      0.0%   7                                 R103D CGTCAGTGGCCTT GACAGCCTCAC GACTCTGCTTCGG                                                                      0.0%   8                                 R103N CGTCAGTGGCCTT AACAGCCTCAC GACTCTGCTTCGG                                                                      0.0%   17                                R103E CGTCAGTGGCCTT GAGAGCCTCAC GACTCTGCTTCGG                                                                      0.0%   18                                R103Q CGTCAGTGGCCTTC AGAGCCTCAC GACTCTGCTTCGG                                                                      0.0%   19                                R103H CGTCAGTGGCCTTC ACAGCCTCAC GACTCTGCTTCGG                                                                      1.7%   20                                R103L CGTCAGTGGCCTTC TCAGCCTCAC GACTCTGCTTCGG                                                                      0.4%   21                                R103K CGTCAGTGGCCTG AAGAGCCTCAC GACTCTGCTTCGG                                                                      25.0%  22                                __________________________________________________________________________

As shown in Table II, mutants having arginine 103 substituted byhistidine (SEQ ID NO: 20) exhibited decreased activity to approximately1.7% of wildtype erythropoietin. Specific activity is again defined aspercent activity of wildtype erythropoietin activity. Mutants havingarginine 103 substituted by leucine (SEQ ID NO: 21) exhibited decreasedactivity to approximately 0.4% of wildtype erythropoietin. Mutantshaving arginine 103 substituted by lysine (SEQ ID NO: 22) exhibiteddecreased activity to approximately 25% of wildtype erythropoietincompared to approximately 10% of wildtype erythropoietin shownpreviously (compare Table I and Table II).

The results show that these three mutant erythropoietin proteins (SEQ IDNOS: 20-22) have some intrinsic agonist activity (biological activity),thus indicating that the erythropoietin muteins (SEQ ID NOS: 20-22) mustbind to the erythropoietin receptor. This phenomenon of weak agonistactivity is commonly seen in pharmacologic blockers when tested at highenough concentrations. Thus, it is reasonable to predict that equivalentquantities of these extremely low activity muteins would competeeffectively with native erythropoietin and block activity.

As shown in Table II, mutants having arginine 103 substituted by alanine(SEQ ID NO: 7), aspartic acid (SEQ ID NO: 8), asparagine (SEQ ID NO:17), glutamic acid (SEQ ID NO: 18), and glutamine (SEQ ID NO: 19)exhibited essentially no erythropoietin biological activity as was shownpreviously (Table I). The results of these experiments indicate thatamino acid position 103 is important for erythropoietin biologicalactivity. Although all of these mutants were expressed and secreted intoculture medium at rates equivalent to that seen for wildtype and othermutants, only very low levels of biological activity were detected or,in some cases, no biological activity was detected. Methods describedherein, such as the ex vivo bioassay of Krystal (Krystal, G., Exp.Hematol. 11:649-660 (1983)), which is an art-recognized bioassay used toevaluate erythropoietin activity, showed that these inactive arginine103 mutants are reduced in activity by at least a 1000-fold below thatof the wildtype human recombinant erythropoietin.

Previously published studies indicated that mutations in the Domain 1region resulted in biologically inactive muteins. (Chern, Y., et al.,Eur. J. Biochem. 202:225-229 (1991)). Thus, modified secretableerythropoietin proteins with mutations in the Domain 1 region would notbe expected to have enhanced biological activity relative to wildtypeerythropoietin proteins. That is, making mutations in this critical andhighly conserved region of the erythropoietin protein would not beexpected to result in the production of muteins with increased specificactivity relative to wildtype erythropoietin proteins. Surprisingly, asshown in Table I, substitution of alanine for serine 100 (SEQ ID NO: 4)and glycine 101 (SEQ ID NO: 5) increased the specific activity of thesemutant proteins.

To determine if the increased specific activity of the muteins obtainedby substitution of alanine for serine 100 (S100A; SEQ ID NO: 4) andglycine 101 (G101A; SEQ ID NO: 5) was statistically significant, astatistical analysis based on the Student-t distribution for smallsamples was performed. The mean values obtained were compared to that ofwildtype erythropoietin activity using the "difference between twosample means" statistic (one-sided). The increased specific activity ofG101A over wildtype was found to be statistically significant at the0.05 level of significance. The increased specific activity of S100A wasnot found to be statistically significant below the 0.010 level ofsignificance.

Additionally, mutants having arginine 14 substituted by alanine (R14A)exhibited decreased activity to approximately 16.4 % of wildtypeerythropoietin. Mutants having arginine 14 substituted by aspartic acid(R14D) exhibited decreased activity to approximately 3.9% of wildtypeerythropoietin.

Structural Integrity of Mutant Erythropoietin Proteins

Previously published studies indicated that mutations in the Domain 1region in which a group of three amino acids was deleted and replacedwith Glu-Phe, caused pronounced structural changes in the molecule.(Chern, Y., et al., Eur. J. Biochem. 202:225-229 (1991)). Thesestructural changes were accompanied by lack of secretion of the mutanterythropoietin from the transfected COS-7 cells. Surprisingly, thisphenomenon was not observed with the more subtle mutations of thepresent invention. Thus, the mutant erythropoietin proteins describedherein provide structurally intact (i.e., with the proper biologicalconformation) mutant erythropoietin proteins.

Assessment of the structural integrity of the mutated erythropoietinproteins of the instant invention was performed by a series ofimmunoprecipitation experiments using anti-peptide monoclonal antibodiesto two domains of the protein, as described in Example 3.

Briefly, the first monoclonal antibody recognizes an epitope withinamino acids 1-26 of erythropoietin. The other monoclonal antibodiesrecognize distinct epitopes within amino acids 99-129. It is known thata gross change in the tertiary structure of erythropoietin would resultin an inability of one or more of the monoclonal antibodies to recognizethe erythropoietin molecule. For example, it has been demonstrated thatradio-iodination of erythropoietin in the presence of chloramine-Tdenatures the molecule, resulting in loss of biological activity andcorresponding loss of recognition by monoclonal antibody.

FIG. 4 shows mutant erythropoietin protein precipitated as percent ofcontrol of wildtype erythropoietin precipitated using three monoclonalantibodies designated across the horizontal axis, 1-26, 99-129α and99-129β. The three erythropoietin proteins examined were the wildtypeerythropoietin, the 103 alanine mutant and the 103 aspartic acid mutant.As seen on the left side of the graph, monoclonal 1-26 recognized eachof the three recombinant erythropoietin proteins with equal efficiency,indicating that mutation of amino acid 103 to either alanine or asparticacid did not result in a gross distortion of erythropoietin'sconformation.

Similarly, as shown in the center of the graph, monoclonal 99-129β alsorecognized the wildtype 103 alanine mutant and 103 aspartic acid mutantwith no statistically significant difference among them. This indicatesthat the conformation within the amino acids 99-129 is similar among thethree recombinant erythropoietin proteins.

Lastly, as shown on the right side of the graph, monoclonal 99-12962recognized both mutant erythropoietin proteins with approximately halfthe efficiency as it recognized the wildtype erythropoietin. This isconsistent with the subtle structural change introduced by a singleamino acid mutation. Taken together, it is reasonable to assume that theinactive point mutants, 103 alanine and 103 aspartic acid, are notgrossly denatured.

Heat Stability of Mutant Erythropoietin Proteins

A previously published study indicated that recombinant humanerythropoietin aggregates as temperature rises. (Endo, Y., et al., J.Biochem. 112(5):700-706 (1992)). Most of the erythropoietin moleculeswithin these multimeric aggregates (twenty erythropoietin molecules peraggregate) would almost certainly not be detectable by antibodies in aradioimmunoassay (RIA). Surprisingly, heat reduced the RIA detection ofwildtype erythropoietin much more rapidly than the more stable mutantsof the present invention. Thus, some of the mutant erythropoietinproteins described herein demonstrate increased heat stability relativeto the wildtype erythropoietin protein.

Assessment of the heat stability of the mutated erythropoietin proteinsof the instant invention was performed by comparing in vitro biologicalactivity with antibody reactivity. Briefly, aliquots of conditionedmedium from erythropoietin cDNA-transfected COS cells were incubated at56° C. for specified time intervals. The samples were cooled on ice anda fraction of each was assessed for biological activity in the Krystalbioassay. The remainder was split into two fractions and erythropoietinprotein was quantified by radioimmunoassay using the commerciallyavailable INCSTAR RIA kit. The results are given in terms of the percentbiological activity remaining or percent protein immunoprecipitatedafter heat treatment compared to untreated samples.

Wildtype erythropoietin exhibits a time-dependent decrease in biologicalactivity when incubated at 56° C. or above (FIG. 5); Tsuda, E., et al.,Eur. J. Biochem. 188:405-411 (1990). Interestingly, a correspondingdecrease in the ability of the commercial radioimmunoassay's antibodiesto recognize this heat-denatured erythropoietin was also observed (FIG.5). This observation was quite reproducible and enabled the use of theRIA to measure the heat stability of the inactive R103A erythropoietincompared to that of wildtype erythropoietin. As seen in FIG. 6A, theheat denaturation curves of R103A and wildtype erythropoietin areessentially identical.

To confirm that this heat stability comparison is sensitive to mutationsin this region of erythropoietin, the effect of the aspartic acidsubstitution (R103D) on the protein's stability was evaluated. Theintroduction of a negatively charged amino acid residue would reasonablybe more structurally disruptive to the molecule than an alanine, andthus be more likely to alter the protein's heat-denaturation curve. Theheat stability of R103D was markedly different (i.e., greater) than thatof wildtype erythropoietin and R103A, as anticipated (FIG. 6B).

To further characterize the nature of the interaction between amino acidresidue 103 and the erythropoietin receptor, site-directed mutagenesiswas used to produce erythropoietin analogs with altered side chainproperties at this position. Arginine was substituted with histidine(R103H), lysine (R103K), asparagine (R103N), glutamine (R103Q), leucine(R103L) and glutamic acid (R103E) to generate 6 new alterederythropoietin molecules. Culture supernatants of cells transfected withthese constructs in a first set of experiments were tested in theKrystal bioassay and the heat stability assay for biological activityand structural stability, respectively.

The heat denaturation curve of R103K was essentially identical to thatgenerated for the wildtype protein. Interestingly, the heat denaturationcurve for R103E was notably different from that of wildtype, and verysimilar to that of R103D. The other 4 mutants had denaturation kineticsintermediate to that of these two proteins. (See FIGS. 6C-6H).

Production of Additional Erythropoietin Proteins Having AlteredBiological Activity

As a result of the identification of sites which are critical toerythropoietin activity in terms of the amino acid residue present andwhich can be altered to produce a mutated sequence which has alteredbiological activity but retains its structural integrity, it is nowpossible to produce modified secretable human recombinant erythropoietinproteins whose ability to regulate the growth and differentiation of redblood cell progenitors is altered (i.e., whose ability to regulate redblood cell progenitors is different from that of the correspondingwildtype human recombinant erythropoietin). These modified humanrecombinant erythropoietin proteins can be secreted in homologous orheterologous expression systems.

As described in the previous sections and in the Examples, such siteshave been identified by oligonucleotide-directed mutagenesis and used tocreate mutant erythropoietin which resulted in substitution of aminoacids at positions 100-109 within Domain 1 (SEQ ID NO: 1), asrepresented in FIG. 1 (SEQ ID NOS: 4-13) and Table I (SEQ ID NOS: 4-16).The data indicate that arginine 103 is critical for erythropoietin'sbiological activity. Additionally, serine 104, leucine 105 and leucine108 appear to play a role, as indicated by the decreased biologicalactivity of these mutants as measured in the above-described bioassays.

It is important to note that the ability of erythropoietin to regulategrowth and differentiation of red blood cell progenitors depends on theability of erythropoietin to bind to its cellular receptor. Importantly,the mutations described herein do not disrupt the structural integrityof the erythropoietin protein, as evidenced by the fact that the mutatedprotein is secreted. That is, as the data presented herein indicate,these mutant erythropoietin proteins retain their biologicalconformation. These results also indicate that Domain 1 amino acids99-110 very likely participate in receptor recognition and activation.

Moreover, as the data presented herein indicate, some mutanterythropoietin proteins also demonstrate increased heat stabilityrelative to the wildtype erythropoietin, even though the biologicalactivity of the mutant has been significantly decreased.

Substitution of alanine at arginine 103 produced erythropoietin mutantswith no detectable erythropoietin activity as measured by standardtechniques. Mutations at serine 104, leucine 105 and leucine 108 alsosignificantly decreased biological activity relative to wildtypeerythropoietin activity. In a similar manner, other changes at one ormore of these critical sites can result in reduction of erythropoietinactivity. Conversely, amino acid residues can be introduced at thesecritical sites to produce modified secretable human recombinanterythropoietin proteins with enhanced biological activity relative towildtype erythropoietin activity.

Conservative substitutions can be made at one or more of the amino acidsites within residues 100-109 of the molecule. For example, alanine andaspartic acid have been used to replace arginine 103. Substitution ofthese amino acids by other amino acids of the same type (i.e., apositively charged, or basic, amino acid for a positively charged, orbasic, one, or an acidic amino acid for an acidic one) as that presentat that specific position can be made and the effect on erythropoietin'sability to regulate the growth and differentiation of red blood cellprogenitors can be determined, using the methods described herein.

Substitutions at these critical sites, alone or in combination, of aminoacids having characteristics different from those of amino acids whosepresence at those sites has been shown to eliminate or reduceerythropoietin activity can also be made and their effect on activityassessed as described above. In particular, substitutions of some, orall, of the amino acids at one, or more, of these critical sites whichresult in modified secretable erythropoietin proteins with enhancederythropoietin activity can be made. Using the techniques describedherein, erythropoietin proteins having enhanced biological activity canbe identified.

In addition, more radical substitutions can be made. For example, anamino acid unlike the residue present in the corresponding position inthe wildtype sequence is substituted for the residue in wildtypeerythropoietin (e.g., a basic amino acid is substituted for an acidicamino acid). Each resulting mutant is then evaluated using theanti-erythropoietin immunoprecipitation techniques and biologicalactivity assays as described.

As a result, modified secretable human recombinant erythropoietinproteins having enhanced erythropoietin activity or increased heatstability can be identified. Similar techniques can be used to identifyadditional critical sites and subsequently, to make substitutions andevaluate their effects on erythropoietin regulating activity.

Applications of Modified Secretable Erythropoietin Proteins HavingAltered Biological Activity

As described above, arginine 103 is essential for erythropoietin'sbiological activity. Additionally, serine 104, leucine 105 and leucine108 also appear to play a significant role in biological activity.Furthermore, these subtle point mutations do not compromise thestructural integrity, (i.e., secretability) of the erythropoietinmolecule. Since these described muteins have some intrinsic biologicalactivity as detected by the assays described herein, albeitsignificantly reduced from wildtype erythropoietin, it is reasonable toassume that they do bind to the erythropoietin receptor. Thus, it isreasonable to assume that the mutant erythropoietin proteins will berecognized by the erythropoietin cellular receptor in essentially thesame manner as the wildtype erythropoietin.

Modified secretable human recombinant erythropoietin proteins of thepresent invention can be used for therapy and diagnosis of varioushematologic conditions. For example, an effective amount of modifiedsecretable recombinant erythropoietin with enhanced biological activityto regulate the growth and differentiation of red blood cell progenitorscan be used therapeutically (in vivo) to treat individuals who areanemic (e.g. as a result of renal disease, chemotherapy, radiationtherapy, or AIDS). An effective amount of modified secretable humanrecombinant erythropoietin protein, as defined herein, is that amount ofmodified secretable erythropoietin protein sufficient to regulate growthand differentiation of red blood cell progenitor cells. For example,modified secretable erythropoietin protein with increased regulatoryability will bind to the erythropoietin receptor and stimulate thegrowth and differentiation of red blood cell progenitor cells. Themodified secretable erythropoietin with enhanced biological activitywould be more potent than the wildtype erythropoietin. Thus, to increasered blood cell growth and differentiation in anemic conditions, a lowereffective dose or less frequent administration to the individual wouldbe required.

Modified secretable erythropoietin with altered regulating activity canalso be used to selectively trigger only certain events regarding thegrowth and differentiation of red blood cell precursors. For example, ithas recently been shown that binding of erythropoietin to its receptorgenerates two distinct chemical signals in cells, a protein kinase Cdependent activation Of the proto-oncogene c-myc and a phosphatasemediated signal to c-myb. (Spangler, R., et al., J. Biol. Chem.266:681-684 (1991); Patel, H. R. and Sytkowski, A. J., Abstract 1205,Blood 78(10) Suppl. 1 (1991)). Thus, a modified secretableerythropoietin can be used to selectively activate either the proteinkinase C or the phosphatase pathways.

An effective amount of modified secretable erythropoietin with decreasedbiological activity relative to wildtype erythropoietin activity, (i.e.,reduced biological activity or no detectable biological activity), canbe used to treat individuals with various erythroleukemias. In thiscase, an effective amount of modified secretable erythropoietin proteinwith decreased regulatory ability will bind to the erythropoietincellular receptor. However, upon the mutant erythropoietin proteinbinding to the receptor, it is reasonable to predict that the mutantprotein lacks ability to trigger subsequent erythropoietin events. It isfurther reasonable to predict that, because the mutant erythropoietindoes bind to the receptor, it prevents wildtype erythropoietin frombinding to the receptor (i.e., competitively inhibits the binding ofwildtype erythropoietin). Thus, the red blood cell progenitors do notproliferate and/or differentiate.

The mutant erythropoietin proteins of the present invention aresecretable, indicating that they retain their structural integrity, andthus fully participate in receptor recognition and binding. The initialinteraction of a hormone with its cognate receptor might be expected toresult in further conformational changes of the hormone ligand, therebystabilizing the hormone/receptor complex and allowing the formation ofhigher ordered complexes. However, if a modified erythropoietin proteinof the present invention, with no detectable erythropoietin activity,binds to its receptor, it is reasonable to assume that the subsequentevents triggered by receptor binding will be altered or inhibited.Therefore, it is also reasonable to assume that growth anddifferentiation of red blood cell progenitor cells will be altered orinhibited, thereby inducing a remission in a red blood cell leukemia.

Recently, a constitutively active (hormone independent) form of themurine erythropoietin receptor was isolated. (Watowich, S. S., Proc.Natl. Acad. Sci. USA 89:2140-2144 (March 1992)). It has also been shownthat the envelope glycoproteins of certain murine viruses bind to andactivate the murine erythropoietin receptor. (Yoshimura, A., Proc. Natl.Acad. Sci. USA 87:4139-4143 (June 1990)). Thus,erythropoietin-independent activation (constitutive activation) of theerythropoietin receptor resulting in red blood cell proliferation in amammal has been demonstrated. It is reasonable to predict that similarconstitutive activation would occur in humans, (for example, a virussimilar to Rauscher or Friend virus) may constitutively activate thehuman erythropoietin receptor also resulting in proliferation of redblood cell progenitors. A modified secretable erythropoietin, whichretains its structural integrity to bind to the receptor, yet does notactivate red blood cell proliferation, would be useful as an antagonistto block such constitutive activation. Moreover, modified secretableerythropoietin proteins with increased stability would providelong-acting erythropoietin antagonists.

Modified secretable erythropoietin would be useful to treat othervarious medical disorders. For example, polycythemia vera ischaracterized by uncontrollable proliferation of red blood cells and iscurrently treated by chemotherapy, radiation or phlebotomy. Theincreased number of red blood cells increases blood viscosity, leadingto a hypertensive condition that can result in a stroke. It isreasonable to predict that an antagonist of erythropoietin, which bindsto the receptor and blocks activation, would be a useful, non-invasivetreatment.

Likewise, individuals with cyanotic heart disease often have ahyper-erythropoietin condition, leading to increased erythrocyteproliferation. Also, renal disease patients that are being treated withwildtype erythropoietin may experience an overdose. Once the wildtypeerythropoietin has been administered, it continues to act. Thus, inthese cases, it would be useful to administer a modified secretableerythropoietin with decreased activity to block the effects of theendogenous and exogenous erythropoietin.

Furthermore, certain hemolytic anemias, such as sickle cell anemia andthalassemia, result in rapid destruction of red blood cells. The bodyresponds by increasing the levels of erythropoietin produced tostimulate red blood cell production. However, the red blood cellsproduced carry defective hemoglobin. It would be useful to use amodified secretable erythropoietin to reduce production of defectiveerythrocytes while another form of therapy is used to stimulate normalhemoglobin synthesis.

Erythropoietin has a relatively short plasma half-life (Spivak, J. L.and Hogans, B. B., Blood 73(1): 90-99 (1989); McMahon, F. G., etal.,Blood 76(9): 1718-1722 (1990)), therefore, therapeutic plasma levelsare rapidly lost, and repeated intravenous administrations must be made.Although the mechanisms responsible for this relatively short plasmahalf-life are not well understood, inactivation due to heatdenaturation/aggregation is likely to play a role. A previouslypublished study indicated that erythropoietin in human serum issusceptible to inactivation by heat. (Elder, G. E., et al., Blood Cells11(3): 409-419 (1986)). Thus, it is reasonable to predict that modifiedsecretable erythropoietin with increased heat stability relative towildtype erythropoietin would have a longer plasma half-life relative towildtype erythropoietin and thus, be useful therapeutically. This may beespecially important in patients with a fever and/or an increasedmetabolic state.

It is also reasonable to predict that modified secretableerythropoietins with enhanced biological activity relative to wildtypeerythropoietin would require a smaller quantity relative to wildtypeerythropoietin to achieve a specified level of biological activity. Thisenhanced biological activity indicates that an effective amount ofmodified erythropoietin with enhanced biological activity issubstantially less than a comparable effective amount of wildtypeerythropoietin. The effective amount of modified erythropoietin withenhanced biological activity is defined herein as the amount of modifiederythropoietin required to elicit an erythropoietin response, asindicated by increased growth and/or differentiation of erythrocyticprecursor cells. Further, the effective amount of modifiederythropoietin with enhanced biological activity will require lessfrequent administration than an equivalent amount of wildtypeerythropoietin. For example, if an effective dose of erythropoietin istypically administered three times a week, modified erythropoietin withenhanced biological activity will only need to be administered once aweek. Thus, a reduced quantity of modified secretable erythropoietinwith enhanced biological activity would be necessary over the course oftreatment than would be necessary if wildtype erythropoietin were used.

Modified secretable erythropoietin may be administered to individualsparenterally or orally. The modified secretable erythropoietin proteinsof this invention can be employed in admixture with conventionalpharmaceutically acceptable carriers. Suitable pharmaceutical carriersinclude, but are not limited to, water, salt solutions and otherphysiologically compatible solutions. The modified secretableerythropoietin proteins of the present invention may be administeredalone, or combined with other therapeutic agents.

It will be appreciated that the amount of modified secretableerythropoietin administered to an individual in a specific case willvary according to the specific modified secretable erythropoietinprotein being utilized, the particular compositions formulated, and themode of application. Dosages for a given individual can be determinedusing conventional considerations such as the severity of the condition,body weight, age and overall health of the individual.

Modified secretable erythropoietin can also be used for diagnosticpurposes. For example, it can be used in assay procedures for detectingthe presence and determining the quantity, if desired, of erythropoietinreceptor. A modified secretable erythropoietin with enhanced activitywould be useful to increase the sensitivity and decrease the incubationtimes of such assays. It can also be used in in vitro binding assays todetermine the effect of new drugs on the binding of erythropoietinprotein to its receptor.

Modified secretable erythropoietin proteins described herein alsoprovide useful research reagents to further elucidate the role oferythropoietin in erythropoiesis, as well as the structure/functionrelationship of erythropoietin and its cellular receptor. For example,modified secretable erythropoietin proteins may be useful for evaluatinga substance for ability to regulate growth and differentiation of redblood cell progenitor cells. A reasonable indication of the ability of asubstance to regulate growth and differentiation of red blood cellprogenitor cells is the extent of binding of the substance to theerythropoietin receptor. The term, extent of binding, as used herein, isdefined to mean the amount of substance bound to the receptor (e.g., thepercent of substance bound to the receptor as compared to a controlsubstance that binds at approximately 100 percent, or alternately, thespecific activity of the test substance). A method for evaluating asubstance for ability to regulate growth and differentiation of redblood cell progenitor cells can comprise comparing the extent of bindingto the erythropoietin receptor of the substance to be evaluated with theextent of binding to the erythropoietin receptor of a modifiedsecretable mutant erythropoietin protein. If the extent of binding tothe erythropoietin receptor of the test substance (i.e., the substanceto be evaluated) is comparable to the extent of binding to theerythropoietin receptor of the modified secretable mutant erythropoietinprotein, then the extent of binding of the test substance is anindication that the ability of the substance to regulate growth anddifferentiation of red blood cell progenitor cells is of approximatelythe same ability as the modified secretable mutant erythropoietin. Forexample, if the specific activity of a test peptide is 25.0%, it isreasonable to assume that the test peptide has the ability to regulategrowth and differentiation of red blood cell progenitor cell comparableto the R103K modified erythropoietin.

The term substance, as used herein, is defined to include proteins,e.g., analogues of wildtype erythropoietin, erythropoietin proteinfragments, other proteins or peptides, and drugs.

The extent of binding to the erythropoietin receptor can be determinedby using any of a number of methods familiar to those of skill in theart. For example, methods such as those described in Yonekura, S. etal., Proc. Natl. Acad. Sci. USA 88:1-5 (1991); Chern, Y. et al., Blood76(11):2204-2209 (1990); and Krystal, G., Exp. Hematol. 11:649-660(1983), the teachings of which are incorporated herein by reference, maybe used.

This invention will now be illustrated by the following Examples, whichare not intended to be limiting in any way.

EXAMPLE 1 Oligonucleotide-Directed Mutagenesis of Human RecombinantErythropoietin

The oligonucleotide-directed mutagenesis used to prepare the modifiedsecretable human recombinant erythropoietin proteins of the presentinvention was performed using the Altered Sites™ In Vitro MutagenesisSystem (Promega Corporation of Madison, Wis.). The Altered Sites™ systemconsists of a unique mutagenesis vector and a simple, straightforwardprocedure for selection of oligonucleotide-directed mutants. The systemis based on the use of a second mutagenic oligonucleotide to conferantibiotic resistance to the mutant DNA strand. The system employs aphagemid vector, pSELECT™-1, which contains two genes for antibioticresistance. One of these genes, for tetracycline resistance, is alwaysfunctional. The other, for ampicillin resistance, has been inactivated.An oligonucleotide is provided which restores ampicillin resistance tothe mutant strand during the mutagenesis reaction. This oligonucleotideis annealed to the single-stranded DNA (ssDNA) template at the same timeas the mutagenic oligonucleotide and subsequent synthesis and ligationof the mutant strand links the two. The DNA is transformed into a repairminus strain E. coli, or other suitable host, and the cells are grown inthe presence of ampicillin, yielding large numbers of colonies. A secondround of transformation in JM109, or a similar host, ensures propersegregation of mutant and wild type plasmids and results in a highproportion of mutants.

The pSELECT-1 plasmid is a phagemid, defined as a chimeric plasmidcontaining the origin of a single-stranded DNA bacteriophage. Thisphagemid produces ssDNA upon infection of the host cells with the helperphage R408 or M13KO7. The vector contains a multiple cloning siteflanked by the SP6 and T7 RNA polymerase promoters and inserted into thelacZ α-peptide. Cloning of a DNA insert into the multiple cloning siteresults in inactivation of the α-peptide. When plated on indicatorplates, colonies containing recombinant plasmids are white in abackground of blue colonies. The SP6 and T7 promoters may be used togenerate high specific activity RNA probes from either strand of theinsert DNA. These sites also serve as convenient priming sites forsequencing of the insert. The pSELECT-1 vector carriers gene sequencesfor both ampicillin and tetracycline resistance. However, the plasmid isampicillin sensitive because a frameshift was introduced into thisresistance gene by removing the Pst I site. Therefore, propagation ofthe plasmid and recombinants is performed under tetracycline selection.

The pSELECT-Control vector provides a convenient white/blue positivecontrol for mutagenesis reactions. This vector was derived from thepSELECT-1 vector by removing the Pst I site within the polylinker. Theresultant frameshift in the lac α-peptide inactivated β-galactosidaseand led to a white colony phenotype on indicator plates. A lacZ repairoligonucleotide (supplied with the system) may be used to introduce afour base insertion which corrects the defect in the lacZ gene andrestores colony color to blue. The fraction of blue colonies obtained isan indication of the mutagenesis efficiency. When the lacZ repairoligonucleotide is used in combination with the ampicillin repairoligonucleotide to correct this defect, 80-90% of the ampicillinresistant colonies are blue. When the lacZ repair oligonucleotide isused alone, a mutagenesis efficiency of only 2-5% is seen.

The mutagenic oligonucleotide must be complementary to thesingle-stranded target DNA. The ssDNA produced by the pSELECT-1 phagemidis complementary to the lacZ coding strand.

The stability of the complex between the oligonucleotide and thetemplate is determined by the base composition of the oligonucleotideand the conditions under which it is annealed. In general, a 17-20 baseoligonucleotide with the mismatch located in the center will besufficient for single base mutations. This gives 8-10 perfectly matchednucleotides on either side of the mismatch. For mutations involving twoor more mismatches, oligonucleotides of 25 bases or longer are needed toallow for 12-15 perfectly matched nucleotides on either side of themismatch.

Routinely, oligonucleotides can be annealed by heating to 70° C. for 5minutes followed by slow cooling to room temperature.

DNA to be mutated is cloned into the pSELECT-1 vector using the multiplecloning sites. The vector DNA is then transformed into competent cellsof JM109, or a similar host, and recombinant colonies are selected byplating on LB plates containing 15 μg/ml tetracycline, 0.5 mM IPTG, and40 μg/ml X-Gal. After incubation for 24 hours at 37° C., coloniescontaining recombinant plasmids will appear white in a background ofblue colonies.

To produce single-stranded template for the mutagenesis reaction,individual colonies containing pSELECT-Control or recombinant pSELECT-1phagemids are grown and the cultures are infected with helper phage asdescribed below. The single-stranded DNA produced is complementary tothe lacZ coding strand and complementary to the strand of the multiplecloning site. Two helper phages R408 and M13KO 7 can be used to providethe greatest latitude in optimizing ssDNA yields.

PROTOCOL

1. Prepare an overnight culture of cells containing pSELECT™-1 orpSELECT™-Control phagemid DNA by picking individual tetracyclineresistant colonies from a fresh plate. Inoculate 1-2 ml of TYP broth(Promega) containing 15 μg/ml tetracycline and shake at 37° C.

2. The next morning inoculate 5 ml of TYP broth containing 15 μg/mltetracycline with 100 μl of the overnight culture. Shake vigorously at37° C. for 30 minutes in a 50 ml tube.

3. Infect the culture with helper phage R408 or M13KO7 at an m.o.i.(multiplicity of infection) of 10 (i.e., add 10 helper phage particlesper cell). For the helper phages supplied with this system, add 40 μl .Continue shaking for 6 hours to overnight with vigorous agitation.

4. Harvest the culture supernatant by pelleting the cells at 12,000 x gfor 15 minutes. Pour the supernatant into a fresh tube and spin againfor 15 minutes.

5. Precipitate the phage by adding 0.25 volume of phage precipitationsolution (Promega) to the supernatant. Leave on ice for 30 minutes, thencentrifuge for 15 minutes at 12,000 x g. Thoroughly drain thesupernatant.

6. Resuspend the pellet in 400 μl of TE buffer (Promega) and transferthe sample to a microcentrifuge tube.

7. Add 0.4 ml of chloroform:isoamyl alcohol (24:1) to lyse the phage,vortex for 1 full minute, and centrifuge in a microcentrifuge (12,000 xg) for 5 minutes. This step removes excess PEG.

8. Transfer the upper, aqueous phase (containing phagemid DNA) to afresh tube, leaving the interface behind. Add 0.4 ml of TE-saturatedphenol:chloroform to the aqueous phase, vortex for 1 full minute, andcentrifuge as in step 7.

9. Transfer the upper, aqueous phase to a fresh tube and repeat thephenol extraction as in step 8. If necessary, repeat this extractionseveral times until there is no visible material at the interface.

10. Transfer the upper, aqueous phase to a fresh tube and add 0.5 volume(200 μl) of 7.5M ammonium acetate plus 2 volumes (1.2 ml) of ethanol.Mix and leave at -20° C. for 30 minutes to precipitate the phagemid DNA.

11. Centrifuge at 12,000 x g for 5 minutes, remove the supernatant,carefully rinse the pellet with 70% ethanol, and centrifuge again for 2minutes. Drain the tube and dry the pellet under vacuum. The pellet maybe difficult to see.

12. Resuspend the DNA in 20 μl of H₂ O. The amount of DNA present can beestimated by agarose gel electrophoresis of a 2 μl sample.

The mutagenesis reaction involves annealing of the ampicillin repairoligonucleotide and the mutagenic oligonucleotide to the ssDNA template,followed by the synthesis of the mutant strand with T4 DNA polymerase.The heteroduplex DNA is then transformed into the repair minus E. colistrain DMH71-18 mutS or other suitable strain. Mutants are selected byovernight growth in the presence of ampicillin. Plasmid DNA is theisolated and transformed into the JM109 strain, or other suitablestrain. Mutant, ampicillin resistant colonies may be screened by directsequencing of the plasmid DNA.

A. Annealing Reaction and Mutant Strand Synthesis

The amount of oligonucleotide required in this reaction may varydepending on the size and amount of the single-stranded DNA template.The ampicillin repair oligonucleotide (27 bases long) should be used ata 5:1 oligo:template ratio and the mutagenic oligonucleotide should beused at a 25:1 oligo:template ratio. A typical reaction may containapproximately 100 ng (0.05 pmol) of ssDNA.

PROTOCOL

1. Prepare the mutagenesis or control annealing reactions as describedbelow.

    ______________________________________                                        Mutagenesis Annealing Reaction                                                1 ssDNAnant pSELECT ™                                                                           0.05 pmol                                                Ampicillin repair oligonucleotide                                                                  1μ (0.25 pmol)                                        (2.2 ng/μl)                                                                Mutagenic oligonucleotide,                                                                         1.25 pmol                                                phosphorylated (see Table 1)                                                  10X Annealing buffer 2 μl                                                  Sterile H.sub.2 O    to final volume 20 μl                                 ______________________________________                                        Control Annealing Reaction                                                    Control ssDNA        100 ng (0.05 pmol)                                       Ampicillin repair oligonucleotide                                                                  1 μl (0.25 pmol)                                      (2.2 ng/μl)                                                                lacZ control oligonucleotide                                                                       1 μl (1.25 pmol)                                      (10.8 ng/gl)                                                                  10X Annealing buffer 2 μl                                                  Sterile H.sub.2 O    to final volume 20 μl                                 ______________________________________                                    

2. Heat the annealing reaction to 70° C. for 5 minutes and allow it tocool slowly to room temperature (15-20 minutes).

3. Place the annealing reaction on ice and add the following:

    ______________________________________                                        10X Synthesis buffer                                                                              3 μl                                                   T4 DNA polymerase (10 u/μl)                                                                    1 μl                                                   T4 DNA ligase (2 u/μl)                                                                         1 μl                                                   Sterile H.sub.2 O   5 μl                                                                       to final volume 20 μl                                  ______________________________________                                    

4. Incubate the reaction at 37° C. for 90 minutes to perform mutantstrand synthesis and ligation.

    ______________________________________                                        Primer Length ng of Primer Equal to 1.25 pmol                                 ______________________________________                                        17 mer        7.0         ng                                                  20 mer        8.3         ng                                                  23 mer        9.5         ng                                                  26 mer        10.8        ng                                                  29 mer        12.0        ng                                                  ______________________________________                                    

B. Transformation into BMH 71-18 mutS

PROTOCOL

1. Add 3 μl of DMSO to 200 μl of BMH71-18 mut S competent cells, mixbriefly, and then add the entire synthesis reaction from step A.4.

2. Let the cells sit on ice for 30 minutes. 3. OPTIONAL: For somestrains, a heat shock at 42° C. for 1-2 minutes after the incubation onice has been reported to increase transformation efficiency. In ourexperience, however, a heat shock does not significantly affect theefficiency of transforming BMH71-18 mut S.

4. Add 4 ml of LB medium and incubate at 37° C. for 1 hour to allow thecells to recover.

5. Add ampicillin to a final concentration of 125 μg/ml and incubate at37° C. for 12-14 hours with shaking.

NOTE: As a control to check the synthesis reaction, 1 ml of the culturecan be removed after the one hour recovery step, spun down, resuspendedin 50 μl of LB medium, and plated on LB plates (pg. 12) containing 125μg/ml ampicillin. This is a check for the presence of ampicillinresistant transformants; a second round of transformation is necessarybefore screening for mutants.

C. Plasmid Mini-Prep Procedure

This procedure is used to isolate pSELECT-1 or pSELECT-Control plasmidDNA from the overnight culture of BMH 71-18 mut S (step B.5, above). Ayield of 1-3 μg of plasmid DNA may be expected.

PROTOCOL

1. Place 1.5 ml of the overnight culture into a microcentrifuge tube andcentrifuge at 12,000 x g for 1 minute. The remainder of the overnightculture can be stored at 4° C.

2. Remove the medium by aspiration, leaving the bacterial pellet as dryas possible.

3. Resuspend the pellet by vortexing in 100 μl of ice-cold minipreplysis buffer (Promega).

4. Incubate for 5 minutes at room temperature.

5. Add 200 μl of a freshly prepared solution containing 0.2N NaOH, 1%SDS. Mix by inversion. DO NOT VORTEX. Incubate for 5 minutes on ice.

6. Add 150 μl of ice-cold potassium acetate solution, pH 4.8 (Promega).Mix by inversion or gentle vortexing for 10 seconds. Incubate for 5minutes on ice.

7. Centrifuge at 12,000 x g for 5 minutes.

8. Transfer the supernatant to a fresh tube, avoiding the whiteprecipitate.

9. Add 1 volume of TE-saturated phenol/chloroform (Promega). Vortex for1 minute and centrifuge at 12,000 x g for 5 minutes.

10. Transfer the upper, aqueous phase to a fresh tube and add 1 volumeof chloroform:isoamyl alcohol)24:1). Vortex for 1 minute and centrifugeas in step 9.

11. Transfer the upper, aqueous phase to a fresh tube and add 2.5volumes of 100% ethanol. Mix and allow to precipitate 5 minutes on dryice.

12. Centrifuge at 12,000 x g for 5 minutes. Rinse the pellet with 70%ethanol (prechilled) and dry the pellet under vacuum.

13. Dissolve the pellet in 50 μl of sterile deionized water. Add 0.5 μlof 100 μg/ml DNase-free RNase A (Promega) and incubate for 5 minutes atroom temperature.

14. The yield of plasmid DNA can be determined by electrophoresis on anagarose gel.

D. Transformation into JM109 Host Cells

PROTOCOL

1. Add 3 μl of DMSO to 200 μl of JM109 competent cells, mix briefly, andadd 0.05-0.10 μg of plasmid DNA from step C.14. Other suitable hostcells may be used.

2. Let the cells sit on ice for 30 minutes.

3. OPTIONAL: A heat shock may be performed at this step.

4. Add 2 ml of LB medium and incubate at 37° C. for 1 hour to allow thecells to recover.

5. Divide the culture into two microcentrifuge tubes and spin for 1minute in a microcentrifuge.

6. Pour off the supernatant and resuspended the cells in each tube in 50μl of LB medium.

7. Plate the cells in each tube on an LB plate containing 125 μg/mlampicillin and incubate at 37° C. for 12-14 hours.

E. Analysis of Transformants

The Altered Sites mutagenesis procedure generally produces greater than50% mutants, so colonies may be screened by direct sequencing. A goodstrategy is to pick 10 colonies and start by sequencing 4 of these. Ifthe mutation is located within 200-300 bases of either end of the DNAinsert, the SP6 or T7 sequencing primers may be used for convenientpriming of the sequencing reactions.

EXAMPLE 2 Cell Culture and Transfection

COS-7 cells were obtained from the American Type Culture Collection(Rockville, Md.) and maintained in Dulbecco's modified Eagle's mediumcontaining 10% fetal bovine serum (GIBCO). Transient expression of cDNAswas performed using a DEAE-Dextran protocol modified by 0.1 mMchloroquine treatment (Sussman, D. J. & Milman, Mol. Cell Biol.4:1641-1645 (1984); Ausubel, F. M., et al., "Current Protocols inMolecular Biology" pp.921-926, John Wiley and Son, New York, (1989)). 3days before the transfection, COS-7 cells were plated at 2×10⁵ /10-cmtissue culture dish. 4 μg DNA were used in each transfection. Medium wascollected 3 days after transfection and assayed for erythropoietinactivity and protein.

EXAMPLE 3 Immunoprecipitation of Erythropoietin

Wildtype and mutant erythropoietin contained in supernatant medium fromCOS cell transfections were diluted one- to four-fold with Dulbecco'smodified Eagle medium containing 10% fetal bovine serum. After one hourincubation at 37 degrees C. with a monoclonal anti-peptide antibody toerythropoietin directed against amino acids 1-26 or 99-129, an equalvolume of Omnisorb (Calbiochem) was added to the samples and thesuspension was incubated for one hour at 4 degrees C. The Omnisorb waspelleted by centrifugation at 4000 rpm for 30 seconds. Theerythropoietin remaining in the supernatant which was not bound by themonoclonal antibody was measured by radioimmunoassay. The amount oferythropoietin bound by antibody (as a percent) was calculated bysubtracting the amount in the supernatant from 100%, the startingconcentration.

Equivalents

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described specifically herein. suchequivalents are intended to be encompassed in the scope of the followingclaims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 22                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ValSerGlyLeuArgSerLeuThrThrLeuLeuArg                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       AspLysThrValSerGlyLeuArgSerLeuThrThrLeuLeuArgAla                              151015                                                                        LeuGly                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GGATAAAGCCGTCAGTGGCCTTCGCAGCCTCACCACTCTGCTTCGGGCTCTGGGAGCC58                  (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 47 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GGATAAAGCCGTCGCTGGCCTTCGCAGCCTCACGACTCTGCTTCGGG47                             (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GCCGTCAGTGCCCTTCGCAGCCTCACGACTCTGCTTCGGG40                                    (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GCCGTCAGTGGCGCTCGCAGCCTCACC27                                                 (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CGTCAGTGGCCTTGCCAGCCTCACGACTCTGCTTCGG37                                       (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 49 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       CCCATACCATAGCGTCAGTGGCCTTGACAGCCTCACGACTCTGCTTCGG49                           (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GGCCTTCGCAGCGCCACGACTCTGCTTCGGG31                                             (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GCCTTCGCAGCCTCGCGACTCTGCTTCGGGC31                                             (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 66 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      CGCAGCCTCACCGCTCTGCTTCGAGCTCTGCGAGCCCCTCACCACTGCGCTTCGAGCTCT60                GGGAGC66                                                                      (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 59 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GCCTCACCACTGCGCTTCGAGCTCTGCGAGCCCCTCACCACTCTGGCTCGGGCTCTGGG59                 (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      CCTCACCACTCTGGCTCGGGCTCTGCG27                                                 (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      GTGGCCTTCGCGCCCTCACGACTCTGCTTC30                                              (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      CCTCACCACTGCGCTTCGAGCTCTGGGAGC30                                              (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      CCTCACCACTCTGGCTCGGGCTCTGGG27                                                 (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      CGTCAGTGGCCTTAACAGCCTCACGACTCTGCTTCGG37                                       (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      CGTCAGTGGCCTTGAGAGCCTCACGACTCTGCTTCGG37                                       (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      CGTCAGTGGCCTTCAGAGCCTCACGACTCTGCTTCGG37                                       (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      CGTCAGTGGCCTGCACAGCCTCACGACTCTGCTTCGG37                                       (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      CGTCAGTGGCCTTCTCAGCCTCACGACTCTGCTTCGG37                                       (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      CGTCAGTGGCCTTAAGAGCCTCACGACTCTGCTTCGG37                                       __________________________________________________________________________

The invention claimed is:
 1. A mutant human erythropoietin proteinhaving an amino acid sequence which differs from the sequence ofwild-type human erythropoietin at the position corresponding toglycine¹⁰¹ of said wild-type erythropoietin, wherein said mutant proteinhas increased biological activity relative to wild-type humanerythropoietin as determined in an in vitro erythropoietic bioassay. 2.A mutant erythropoietin protein according to claim 1, wherein the aminoacid residue at the position corresponding to glycine¹⁰¹ of wild-typeerythropoietin is alanine.
 3. -human erythropoietin.
 4. A nucleic acidmolecule encoding a mutant human erythropoietin protein according toclaim
 1. 5. A nucleic acid molecule encoding a mutant humanerythropoietin protein according to claim
 2. 6. A nucleic acid moleculeencoding -human erythropoietin according to claim
 3. 7. A pharmaceuticalcomposition comprising a mutant human erythropoietin protein accordingto claim 1 and a pharmaceutically acceptable carrier.
 8. Apharmaceutical composition comprising a mutant human erythropoietinprotein according to claim 2 and a pharmaceutically acceptable carrier.9. A pharmaceutical composition comprising -human erythropoietinaccording to claim 3 and a pharmaceutically acceptable carrier.
 10. Amethod of promoting the growth and differentiation of red blood cellprogenitors in an individual, comprising the step of administering amutant human erythropoietin protein according to claim 1 to saidindividual.
 11. A method of promoting the growth and differentiation ofred blood cell progenitors in an individual, comprising the step ofadministering a mutant human erythropoietin protein according to claim 2to said individual.
 12. A method of promoting the growth anddifferentiation of red blood cell progenitors in an individual,comprising the step of administering -human erythropoietin according toclaim 3 to said individual.
 13. A mutant human erythropoietin proteinhaving an amino acid sequence which differs from the sequence ofwild-type human erythropoietin at the position corresponding toarginine¹⁰³ of said wild-type erythropoietin, wherein said mutantprotein has decreased biological activity relative to wild-type humanerythropoietin as determined in an in vitro erythropoietic bioassay, andwherein a recombinant host cell producing a polypeptide having the aminoacid sequence of said mutant protein is capable of secreting saidpolypeptide.
 14. A mutant erythropoietin protein according to claim 13,wherein said mutant protein has increased heat stability relative towild-type erythropoietin.
 15. A mutant erythropoietin protein accordingto claim 13, wherein the amino acid residue at the positioncorresponding to arginine¹⁰³ of wild-type erythropoietin is aspartate.16. -human erythropoietin.
 17. A nucleic acid molecule encoding a mutanthuman erythropoietin protein according to claim
 13. 18. A nucleic acidmolecule encoding a mutant human erythropoietin protein according toclaim
 14. 19. A nucleic acid molecule encoding a mutant humanerythropoietin protein according to claim
 15. 20. A nucleic acidmolecule encoding -human erythropoietin according to claim
 16. 21. Apharmaceutical composition comprising a mutant human erythropoietinprotein according to claim 13 and a pharmaceutically acceptable carrier.22. A pharmaceutical composition comprising a mutant humanerythropoietin protein according to claim 14 and a pharmaceuticallyacceptable carrier.
 23. A pharmaceutical composition comprising a mutanthuman erythropoietin protein according to claim 15 and apharmaceutically acceptable carrier.
 24. A pharmaceutical compositioncomprising -human erythropoietin according to claim 16 and apharmaceutically acceptable carrier.
 25. A method of inhibiting thegrowth and differentiation of red blood cell progenitors in anindividual, comprising the step of administering a mutant humanerythropoietin protein according to claim 13 to said individual.
 26. Amethod of inhibiting the growth and differentiation of red blood cellprogenitors in an individual, comprising the step of administering amutant human erythropoietin protein according to claim 14 to saidindividual.
 27. A method of inhibiting the growth and differentiation ofred blood cell progenitors in an individual, comprising the step ofadministering a mutant human erythropoietin protein according to claim15 is to said individual.
 28. A method of inhibiting the growth anddifferentiation of red blood cell progenitors in an individual,comprising the step of administering -human erythropoietin according toclaim 16 to said individual.